This research involves a combined cytogenetic, somatic cell genetic, immunological and molecular approach to the role of mammalian ribosomal RNA gene amplification in carcinogenesis and tumor progression. Q-, G-, and R-banding will be used to identify probable amplified regions in tumor cell lines and normal cells. AgNOR staining to detect actively transcribed rRNA genes, antibodies to 5-methyl-cytidine and an indirect immunoperoxidase technique to detect chromosome regions highly enriched in 5-methylcytosine. In situ hybridization to radiolabeled 18S and Z8S rRNA probes will show the location and multiplicity of these coding sequences in chromosome segments and in Southern blots of restriction endonuclease fragments. Hpa II and Msp I digestion of amplified hypermethylated DNA will be used, in conjunction with digestion by other enzymes, to map the sites of methylation that are potentially important in rRNA gene regulation. Attention will be focused on hypermethylated, amplified and transcriptionally inactive rRNA genes in the form of HSRs and DMs. We will attempt to activate them by demethylation with 5-azacytidine in order to establish the role of DNA methylation in rRNA gene regulation. Restriction mapping of the rDNA of reactivated genes may clarify the sites of methylation involved in rRNA gene regulation. The relationship between amplification and hypermethylation of rRNA genes will be studied in various ways. The nature of the amplified sequences will be studied using cloned fragments of the amplified sequences. We will study the expression and methylation of cloned mouse and human rDNA fragments that have been experimentally amplified by cotransformation into DHFR-hamster cells with cloned DHFR DNA and selection for methotrexate resistant sublines. Cotransformation and coamplification of specific rDNA fragments will also be used to determine the boundaries of the region essential for organizing silver-staining chromatin. (A)